thyroid hormone regulates the expression of the sonic...

Endocrinology 2011 152:1989-2000 originally published online Mar 1, 2011; , doi: 10.1210/en.2010-1396

Shubha Tole and Vidita A. Vaidya Lynette A. Desouza, Malini Sathanoori, Richa Kapoor, Neha Rajadhyaksha, Luis E. Gonzalez, Andreas H. Kottmann,

Pathway in the Embryonic and Adult Mammalian Brain

Thyroid Hormone Regulates the Expression of the Sonic Hedgehog Signaling

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Thyroid Hormone Regulates the Expression of theSonic Hedgehog Signaling Pathway in the Embryonicand Adult Mammalian Brain

Lynette A. Desouza, Malini Sathanoori, Richa Kapoor, Neha Rajadhyaksha,Luis E. Gonzalez, Andreas H. Kottmann, Shubha Tole, and Vidita A. Vaidya

Department of Biological Sciences (L.A.D., M.S., R.K., N.R., S.T., V.A.V.), Tata Institute of FundamentalResearch, Mumbai 400005, India; and Center for Motor Neuron Biology and Disease and ColumbiaGenome Center (L.E.G., A.H.K.), Columbia University, College of Physicians and Surgeons, New York,New York 10032

Thyroid hormone is important for development and plasticity in the immature and adult mammalianbrain. Several thyroid hormone-responsive genes are regulated during specific developmental timewindows, with relatively few influenced across the lifespan. We provide novel evidence that thyroidhormone regulates expression of the key developmental morphogen sonic hedgehog (Shh), and itscoreceptorspatched(Ptc)andsmoothened(Smo), intheearlyembryonicandadultforebrain.Maternalhypo- and hyperthyroidism bidirectionally influenced Shh mRNA in embryonic forebrain signalingcenters at stages before fetal thyroid hormone synthesis. Further, Smo and Ptc expression were sig-nificantly decreased in the forebrain of embryos derived from hypothyroid dams. Adult-onset thyroidhormoneperturbationsalsoregulatedexpressionoftheShhpathwaybidirectionally,withasignificantinduction of Shh, Ptc, and Smo after hyperthyroidism and a decline in Smo expression in the hypothy-roid brain. Short-term T3 administration resulted in a significant induction of cortical Shh mRNA ex-pression and also enhanced reporter gene expression in Shh�/LacZ mice. Further, acute T3 treatment ofcortical neuronal cultures resulted in a rapid and significant increase in Shh mRNA, suggesting directeffects. Chromatin immunoprecipitation assays performed on adult neocortex indicated enhancedhistone acetylation at the Shh promoter after acute T3 administration, providing further support thatShh is a thyroid hormone-responsive gene. Our results indicate that maternal and adult-onset pertur-bations of euthyroid status cause robust and region-specific changes in the Shh pathway in the em-bryonic and adult forebrain, implicating Shh as a possible mechanistic link for specific neurodevelop-mental effects of thyroid hormone. (Endocrinology 152: 1989–2000, 2011)

Thyroid hormone deficiencies during critical periods ofbrain development result in profound structural as

well as functional abnormalities (1–4). Several studiessuggest that the critical period in which thyroid hormoneexerts dramatic effects on neuronal structure appears to bepredominantly during early postnatal life (1). However,maternally derived thyroid hormone, transported via theplacenta, is known to alter neural progenitor prolifera-tion, differentiation, and migration within the developingembryo, which expresses thyroid hormone receptor (TR)isoforms before the onset of fetal thyroid hormone syn-

thesis (5–11). Furthermore, thyroid hormone is known toexert effects on neuronal and glial progenitor prolifera-tion, survival, and maturation within the adult brain (12–18). Clinical evidence indicates that early gestational de-privation of thyroid hormone, as well as adult-onsethypothyroid status, result in cognitive and functional neu-rological impairments, with the early onset effects associ-ated with more severe consequences (1, 3, 19). Taken to-gether, these studies provide evidence of a role for thyroid

ISSN Print 0013-7227 ISSN Online 1945-7170Printed in U.S.A.Copyright © 2011 by The Endocrine Societydoi: 10.1210/en.2010-1396 Received December 6, 2010. Accepted February 11, 2011.First Published Online March 1, 2011

Abbreviations: AcH3, Acetylated H3; AcH4, acetylated H4; ChIP, chromatin immunopre-cipitation; CT, threshold cycle; DG, dentate gyrus; e, embryonic day; GFAP, glial fibrillaryacidic protein; H3, histone 3; H4, histone 4; HPRT, hypoxanthine phosphoribosyl transferase;Hyp, hypothalamus; MAP, microtubule-associated protein; MMI, 2-mercapto-1-methylimida-zole; NeuN, neuronal nuclei; Ptc, patched; PTU, 6-n-propyl-2-thiouracil; qPCR, quantitativePCR; Shh, sonic hedgehog; Smo, smoothened; SVZ, subventricular zone; TR, thyroid hormonereceptor; TRE, thyroid hormone response element; VDB, vertical limb of the diagonal band; VT,ventral telecephalon; Xhh, Xenopus hedgehog; Zli, zona limitans intrathalamica.

N E U R O E N D O C R I N O L O G Y

Endocrinology, May 2011, 152(5):1989–2000 endo.endojournals.org 1989

hormone in modulating structure and function in themammalian brain throughout life.

Thyroid hormone effects are mediated through thetranscriptional regulation of target genes via distinct TRisoforms, encoded by the TR� and TR� genes (20). Severalthyroid hormone-responsive genes, including laminin,transient axonal glycoprotein-1, reelin, neurogenic differ-entiation, rabconnectin 3/neurogranin, and Dab-1, havebeen implicated in mediating thyroid hormone effects onneurodevelopment (21–24). Although many thyroid hor-mone-responsive genes are sensitive to thyroid hormoneperturbations only during critical periods, there are rela-tively fewer target genes that are regulated by thyroid hor-mone across the lifespan (25–27). Given that thyroid hor-mone influences neural and glial progenitors in both thedeveloping as well as mature nervous system, we hypoth-esized that the expression of developmental morphogens,which retain a powerful influence on these progenitorsacross the lifespan, may be regulated by thyroid hormone.Sonic hedgehog (Shh), a major developmental morpho-gen, is thought to be a key regulator of neural and oligo-dendroglial progenitors across development and intoadulthood (28–31). Shh mediates its biological effects viaits membrane-associated receptors smoothened (Smo) andpatched (Ptc, Ptch1), such that Shh binding to Ptc relievesthe inhibitory influence of Ptc on Smo, thus activating thedownstream cascade (32). Strikingly, during amphibianmetamorphosis, thyroid hormone is reported to exert itsmetamorphic effects within the gastrointestinal system viainfluencing Xenopus hedgehog (Xhh) expression, indicat-ing that this developmental signaling pathway can be tar-geted by thyroid hormone in vertebrates (33). We hypoth-esized that perturbations of thyroid hormone status mayinfluence expression of Shh, or its receptors Ptc and Smo,both in the embryonic and adult mammalian brain. Here,we provide novel evidence that perturbations of maternaland adult thyroid hormone status results in a robust andbidirectional regulation of the expression of the Shh sig-naling cascade in the embryonic and adult rodent brain,respectively, suggesting that this pathway may mediatesome of the important neurodevelopmental effects of thy-roid hormone perturbations.

Materials and Methods

Animal treatment paradigmsSprague Dawley rats (220–270 g) bred in our animal-breed-

ing colony were used for all experiments. Animals were grouphoused and maintained on a 12-h light, 12-h dark cycle withaccess to food and water ad libitum. All animal procedures werecarried out in accordance with the National Institutes of HealthGuide for the Care and Use of Laboratory Animals and approvedby the Tata Institute of Fundamental Research and Columbia Uni-

versity Institutional Animal Ethics Committees. Shh�/LacZ miceweregeneratedbyA.Kottmannthrough the targetingofan internalribosome entry site LacZ construct into the 3� untranslated regionof the Shh gene keeping the Shh coding sequence intact (34, 35).

To address the influence of maternal hypothyroidism on theShh signaling cascade in the embryo, female rats were subjectedto sham surgery or thyroidectomized (n � 4–5 dams per group)and allowed recovery for a week. Thyroidectomized and controldams were set up for mating with the day of vaginal plug beingconsidered as d 0.5. After detection of a vaginal plug, 6-n-propyl-2-thiouracil (PTU) (0.05%; Sigma, St. Louis, MO) was added tothe drinking water of thyroidectomized dams till sacrifice. Em-bryos were harvested from hypothyroid and control animals atembryonic d (e)13.5 (n � 11–15 embryos per group). All em-bryos used in these studies were age matched. Embryos harvestedfrom hypothyroid dams showed decreased body size, and to ruleout differences due to a possible developmental lag, all embryosused in the study were also somite matched. To address the in-fluence of maternal hyperthyroidism, pregnant dams received T3

(1 mg/kg) or vehicle (0.02 N NaOH) injections ip from the day thevaginal plug was observed until killing at e13.5 (n � 7 embryosper group derived from three to four dams per group). Afterdecapitation, the embryonic head was frozen on dry ice andstored at �70 C.

To address the influence of adult-onset hypothyroidism onShh pathway expression, male rats received the goitrogens2-mercapto-1-methylimidazole (MMI) (Sigma) or PTU (n �4/group). MMI (0.025%) or PTU (0.05%) in drinking water fora period of 28 and 21 d, respectively, whereas controls receivednormal drinking water. To address the influence of adult-onsethyperthyroidism, T3 (0.5 mg/kg) or vehicle (0.02 N NaOH) wassc administered daily for 10 d with animals killed 2 h after the lastT3 injection (n � 5–6/group). To address the influence of acuteT3 treatment, adult male rats received a single sc injection of T3

(0.5 mg/kg) or vehicle (0.02 N NaOH) and were killed 3 h later[n � 3–5/group for in situ hybridization and n � 7–10/group forchromatin immunoprecipitation (ChIP)]. All animals were killedvia rapid decapitation, and brains were fresh frozen on dry iceand stored at �70 C until use.

To examine the influence of short-duration thyroid hormonetreatment, Shh�/LacZ reporter mice were injected sc twice dailywith T3 (0.5 mg/kg) or vehicle (0.02 N NaOH) for 2 d and killed2 d after the last T3 injection (n � 5/group) via transcardialperfusion. The brains were postfixed in 4% paraformaldehydeand free-floating sections (30 �M) obtained using a vibratome(TPI, St. Louis, MO).

Trunk blood was collected from all animals at the time ofkilling and serum T3 levels determined using the commerciallyavailable RIA kit (RIAK-4/4A; BRIT, Mumbai, India) as previ-ously described (36). Serum T3 levels are shown in SupplementalTable 1, published on The Endocrine Society’s Journals Onlineweb site at http://endo.endojournals.org.

Primary cortical culturesCortical neurons were isolated from rat embryos (e17.5). Af-

ter removal of meninges, the cleaned cortices were placed intrypsin-EDTA (Invitrogen, Carlsbad, CA) for 15 min followedby washes with cold HEPES buffered Hank’s balanced salt so-lution (Invitrogen) and dissociated in culture medium (Neuro-basal medium supplemented with 2% B27 supplement, 0.5 mM

L-glutamine, 5 U/ml penicillin, and 5 U/ml streptomycin) (Invit-rogen). Cells were plated on Poly-D-lysine (Sigma) coated 35-mm

1990 Desouza et al. T3 Regulates Shh Cascade Expression Endocrinology, May 2011, 152(5):1989–2000

dishes at a density of 106 cells/dish. Neurons were allowed toattach and extend processes for 9 d in vitro before initiatingtreatment with 20 nM T3 (Sigma) for 3 h. Cells were harvested 3 hafter T3 treatment and processed for RNA extraction, with allassays performed in triplicate.

Quantitative PCR (qPCR)RNA purification, cDNA synthesis, and qPCR were performed

as described previously (37). In brief, total RNA was isolated usingTriReagent (Sigma),according to themanufacturer’sprotocol.TheRNA was quantified using Nanodrop (Eppendorf, Hamburg, Ger-many) and 2 �g of RNA per sample was used to prepare cDNAusing the Quantitect RT kit (QIAGEN, Valencia, CA). cDNA wasamplified in a Realplex mastercycler (Eppendorf) and visualizedusing a SYBR Green kit (Applied Biosystems, Foster City, CA).Hypoxanthine phosphoribosyl transferase (Hprt) was used as anendogenoushousekeepinggenecontrol.Tocomparetheexpressionof Hprt and target genes, the comparative threshold cycle (CT)method was used as described previously (38). �CT � absolute CT

value � endogenous CT value; and ��CT � �CT T3 treatment ��CT control. Data are fold change � SEM compared with control.Primer sequences used are described in Supplemental Table 2.

In situ hybridizationIn situ hybridization was carried out as previously described

(39, 40). Cryostat-cut sections were thaw mounted ontoProbe-on plus slides (Electron Microscopy Sciences, Hatfield,PA). Slides were fixed, acetylated, and dehydrated before storageat �70 C. Smo, Ptc, and Shh cRNA probes were generated fromtranscription competent pGEM-4Z plasmids provided by Mar-tial Ruat (Centre National de la Recherche Scientifique, France).Antisense cRNA probes were transcribed using 35S-labeled uri-dine-5�-triphosphate (Amersham, Buckinghamshire, UK). Slideswere incubated for 18–20 h at 60 C in hybridization buffercontaining 35S-uridine-5�-triphosphate-labeled riboprobes at aconcentration of 1 � 106 cpm/150 �l. Within an individual ex-periment, all slides were exposed to the same batch of riboprobe.After hybridization, slides were subjected to ribonuclease A (20�g/ml) treatment, followed by stringent washes in decreasing con-centrations of saline sodium citrate. Slides were air dried and ex-posed to Biomax film (Kodak, New York, NY) for 3 wk. All slideswithin a single experiment were exposed to the same film to reducevariability that may arise during autoradiographic film develop-ment. Sense riboprobes, or a ribonuclease (40 �g/ml at 37 C for 30min) prehybridization wash, did not yield significant hybridization(data not shown), confirming the specificity of the signal observed.

Levels of Shh, Ptc, and Smo transcripts were analyzed usingthe Macintosh-based Scion Image software (Scion, Frederick,MD). To correct for nonlinearity, 14C standards were used forcalibration. An equivalent area was outlined for each sample,and optical density measurements (6–8) from both sides of threeto four individual sections from each animal were analyzed, fromwhich the mean value was calculated. Shh mRNA expression inembryonic brain was quantitated in the ventral telecephalon(VT), zona limitans intrathalamica (Zli), and hypothalamus(Hyp). Shh transcript expression in the adult brain was analyzedin the vertical limb of the diagonal band (VDB), cingulate cortex,cortical layer V, medial region and lateral region of striatum, anddentate gyrus (DG) subfield of the hippocampus. Smo and PtcmRNA expression was determined in the embryonic brainwithin the VT, Zli, Hyp, and cortex. Smo and Ptc mRNA ex-

pression was determined in the adult brain within the cortex,medial and lateral striatum, and subventricular zone (SVZ) lin-ing the lateral ventricles and the DG.

ImmunofluorescenceImmunofluorescence staining for �-galactosidase was per-

formed on sections from Shh�/LacZ animals. In brief, sections wereincubated with primary antibody: goat anti-�-galactosidase (1:250; AbD Serotec, Kidlington, UK) for 3 d at 4 C and were thenincubated with a secondary antibody: Alexa Fluor 488-conjugatedantigoat (1:250; Invitrogen) for 2 h. Quantitative analysis for �-ga-lactosidase immunopositive cells was performed by an experi-menter blind to the treatment conditions, and only those cells thatwere strongly immunopositive were counted as �-galactosidasepositive cells. Double immunofluorescence experiments for �-ga-lactosidase with the neuronal marker neuronal nuclei (NeuN), thechondroitin sulfate proteoglycan NG2, the 2�,3�-cyclic nucleotide3�-phosphodiesterase RIP, or the glial fibrillary acidic protein(GFAP) were carried out as previously described (35). In brief,sections were incubated with primary antibody cocktails of goatanti-�-galactosidase with mouse anti-NeuN (1:500; MilliporeCorp., Bedford, MA) or mouse anti-RIP (1:10; DevelopmentalStudies Hybridoma Bank, Iowa City, IA) along with rabbit anti-NG2 (1:250; Millipore Corp.) or rabbit anti-GFAP (1:250; Milli-pore Corp.). After incubation with primary antibody for 3 d at 4 C,sections were washed and incubated with a cocktail of secondaryantibodies: Alexa Fluor 488-conjugated antigoat (1:250); rhod-amine-conjugated antimouse IgG (1:500; Millipore Corp.), andCy-5-conjugated antirabbit IgG (1:500; Millipore Corp.) for 2 h.Sections were mounted onto slides with Vectashield (Vector Lab-oratories, Burlingame, CA) and analyzed using a Zeiss Axioplan2confocal laser scanning microscope (Zeiss, Oberkochen, Ger-many). Double immunofluorescence experiments on primary cor-tical neurons for all TR antibodies with the neuronal marker mi-crotubule-associated protein (MAP)-2 were carried out asdescribed above using a combination of all the TR antibodies, i.e.goat anti-TR�, TR�1, and TR�2 (1:200 each; Santa Cruz Biotech-nology, Inc., Santa Cruz, CA) along with mouse anti-MAP-2 (1:1000; Sigma). Secondary antibodies used were Alexa Fluor 488-conjugated antigoat (1:400) and rhodamine-conjugated antimouseIgG (1:500). Nuclei were counterstained using Hoechst 33342(Invitrogen).

ChIP assayChIP was carried out as described previously (41). Briefly,

bilateral cortices were dissected, fixed to cross-link the DNA andthe bound proteins. The tissue was dounce homogenized, soni-cated, and immunoprecipitated using a pan-acetylation histone3 (H3) or pan-acetylation histone 4 (H4) antibody (1 �g; CellSignaling Technology, Beverly, MA). After reverse cross-linkingand chromatin precipitation, qPCR analysis was performedwithin upstream regions of the Shh gene. Putative TR bindingsites were analyzed for the 5� upstream sequence of the rat Shhgene from �6500 to the transcriptional start site using AliBaba2.1 (http://www.gene-regulation.com/pub/programs.html). Ofthe several putative TR� and TR� binding sites identified, aregion containing both putative TR� and TR� binding sites wasamplified, and a second region from �184 to the transcriptionalstart site was amplified. We also performed qPCR analysis toexamine possible enrichment of acetylated histones H3 (AcH3)and H4 (AcH4) within upstream regions of the Ptc and Smo


http://www.gene-regulation.com/pub/programs.html

genes. In each sample, the results were normalized to a regionamplified from the glyceraldehyde-3-phosphate dehydrogenasepromoter. Primer sequences used in ChIP experiments are de-scribed in Supplemental Table 2.

Statistical analysisResults were subjected to statistical analysis using Student’s

unpaired t test for experiments with two groups and one-wayANOVA for experiments with three groups (Prism; GraphPad,San Diego, CA). When two groups compared exhibited unequalvariances, statistical analysis was performed using a Student’sunpaired t test with a Welch correction. Differences were con-sidered to be statistically significant at P � 0.05.

Results

Maternal hypothyroidismdecreases Shh signaling cascadeexpression in the embryonic ratbrain

Shh is expressed in ventral signalingareas within the Hyp and ventral telen-cephalon, where it is critical for the de-velopment of the ventral forebrain, spec-ifying cell fate choice, proliferation, andsurvival (42–44). Shh is also expressedwithin the Zli, where it regulates the de-velopment of adjacent dorsal and ventralthalamic structures (45–48). Althoughmost studies have focused on the conse-quencesofhypothyroidismat lateembry-onic and postnatal stages in the rodentbrain, the fetus that expresses TRs beforethe onset of fetal thyroid hormone syn-thesis is exposed to maternal thyroid hor-mone transported via the placenta fromthe earliest stages of embryonic forebraindevelopment. We analyzed the conse-quences of maternal hypothyroidism onthe regulation of Shh mRNA and its re-ceptors in the early embryonic brain.E13.5 embryos harvested from hypothy-roid dams exhibited a significant down-regulation of Shh mRNA in the Zli (Fig.1B). Expression of Shh in the ventral tel-encephalon and the Hyp remained un-changed in these embryos. In addition,Ptc mRNA expression in the embryonicbrain was also significantly down-regu-lated in the ventral telencephalon and inthe Zli (Fig. 1C). The Shh signaling recep-tor, Smo, displayed a down-regulation ina more wide-spread manner, in areas thatare responsive to secreted Shh, includingthe entire thalamus, the ventral telen-cephalon, and the developing embryonic

neocortex (Fig. 1D). Our results indicate that maternal hy-pothyroidism significantly decreases expression of Shh andits coreceptors Ptc and Smo in the embryonic rodent brain.

Maternal hyperthyroidism enhances theexpression of Shh in the embryonic rat brain

In contrast to the decreased Shh ligand expression inembryos derived from hypothyroid dams, an opposite ef-fect was seen in embryos obtained from hyperthyroiddams. These embryos exhibited a significant up-regula-tion in Shh mRNA levels in the major signaling centers of

FIG. 1. Maternal hypothyroidism decreases Shh signaling cascade expression in theembryonic rat brain. Rat e13.5 pups were obtained from thyroidectomized females that wereadministered PTU (Tx � PTU), and levels of Shh, Ptc, and Smo mRNA were determined by insitu hybridization. Shown is a schematic representation of a coronal section through theembryonic rat brain with a color code depicting the regions quantitated (A) andrepresentative autoradiographs of Shh (B), Ptc (C), and Smo (D) mRNA in embryos derivedfrom control and hypothyroid (Tx � PTU) female dams. Maternal hypothyroidism resulted in asignificant decrease in Shh mRNA in the Zli with no change in the VT or Hyp (B). Ptc and SmomRNA were significantly down-regulated in several forebrain regions (namely the VT, Zli,Lat.Ctx and Med.Ctx) of e13.5 embryos derived from hypothyroid dams (C and D). Results areexpressed as a percentage of control and are the mean � SEM (n � 11–15 embryos per groupderived from four to five dams in each group). *, P � 0.05 compared with embryos derivedfrom control dams (Student’s unpaired t test). White bars, Embryos derived from vehicle-treated dams; black bars, embryos derived from Tx � PTU-treated dams.


the ventral telencephalon, Zli, and in the hypothalamicregion (Fig. 2A). However, we did not observe any changein the expression of the Shh receptors, Ptc and Smo (Fig.2, B and C). Our results indicate that Shh signaling fromembryonic ventral forebrain signaling centers is likely tobe enhanced with maternal hyperthyroidism due to a ro-bust up-regulation of the ligand.

Adult-onset hypothyroid status selectivelydecreases Smo mRNA in the DG subfield ofthe hippocampus

Shh continues to be expressed within specific regions inthe adult mammalian brain, including the neocortex, stria-tum, and the VDB (49). Although Ptc expression is wide-spread in the adult brain, the signaling coreceptor Smo ispredominantly expressed in the adult neurogenic niches,namely the SVZ lining the lateral ventricles and in the DGsubfield of the hippocampus (49). Adult hypothyroid sta-tus resulted in no change in the expression of Shh mRNA(Supplemental Fig. 1). The expression of Ptc mRNA aswell remained unaltered in adult hypothyroid animals

(Supplemental Fig. 1). In contrast, adult-onset hypothy-roidism significantly down-regulated the expression of thesignaling receptor Smo in the DG (Fig. 3B). Interestingly,hypothyroid animals did not show any change in Smoexpression within the other major neurogenic niche of theSVZ (Fig. 3B).

Shh mRNA is significantly up-regulated in theadult rat brain in response to chronic T3

administrationChronic T3 administration in adulthood resulted in a

significant up-regulation of Shh mRNA in several brainregions, including the cortex, VDB, and lateral striatum(Fig. 4, B and C). Further, Ptc mRNA expression was alsosignificantly up-regulated in several regions of the adultrodent brain (Fig. 4, B and D). In addition, mRNA ex-pression of the Shh signaling receptor Smo was also in-creased in the DG (Fig. 4, B and E). Strikingly, Smo ex-pression was also robustly enhanced in superficial layersof neocortex in hyperthyroid brains, whereas Smo mRNAsignal was almost undetectable in euthyroid controls usingin situ hybridization (Fig. 4, B and E).

FIG. 2. Maternal hyperthyroidism enhances the expression of Shh inthe embryonic rat brain. Rat e13.5 pups were derived from vehicle-treated and chronic T3-treated dams, and the levels of Shh, Ptc, andSmo mRNA were determined by in situ hybridization. Shown arerepresentative autoradiographs showing Shh mRNA expression (A) inthe VT, Zli, and Hyp in e13.5 embryos derived from vehicle or T3-treated dams. Maternal hyperthyroidism resulted in a significantinduction in Shh mRNA in the VT, Zli, as well as Hyp, of the embryonicbrain (A). Ptc and Smo mRNA remained unaltered in embryos derivedfrom maternally hyperthyroid dams (B and C). Results are expressed asa percentage of control and are the mean � SEM (n � 7/group derivedfrom three to four dams in each group). *, P � 0.05 when comparedwith control (Student’s unpaired t test). White bars, embryos derivedfrom vehicle-treated dams; black bars, embryos derived from chronicT3-treated dams.

020

40

60

80100120140

Smo

mR

NA

(% C

ontro

l)

**

SVZ DG

PTU

M

MI

C

ontro

l

A

Dentate Gyrus (DG)

B

Subventricular Zone (SVZ)

FIG. 3. Adult-onset hypothyroidism decreases Smo mRNA expressionin the DG subfield of the hippocampus. Adult male rats were renderedhypothyroid by administration of MMI for 28 d, or PTU for 21 d, andthe levels of Smo mRNA were determined by in situ hybridization.Shown are schematic representations of coronal sections through theventricular zone and hippocampus of the adult rodent brainhighlighting the SVZ and the DG region of the hippocampus,respectively (A). Smo mRNA was significantly and selectively down-regulated in the DG after adult-onset hypothyroidism compared withvehicle-treated controls (B). Results are expressed as a percentage ofcontrol and are the mean � SEM (n � 4/group). *, P � 0.05 comparedwith control (one-way ANOVA, Bonferroni post hoc test). White bars,Vehicle-treated controls; gray bars, MMI-treated animals; black bars,PTU-treated animals.


Subchronic T3 administration enhances neocortical�-galactosidase positive cell numbers in Shh�/LacZ

miceShort-duration T3 treatment in Shh�/LacZ mice resulted

in a significant increase in the number of �-galactosidaseimmunopositive cells within the neocortex (Fig. 5A).These results are consistent with the hypothesis that Shh isa thyroid hormone-responsive gene in the adult mamma-lian brain. To gain an understanding of the cell types thatexpress Shh within the neocortex (Fig. 5B), we performeddouble labeling experiments for �-galactosidase with neu-ronal and glial markers. Cells expressing �-galactosidasecolocalized with the neuronal marker NeuN (Fig. 5C),indicating presence of the reporter in cortical neurons. We

did not observe any �-galactosidase ex-pression within oligodendroglial cellsexpressing NG2 or RIP or in GFAP-im-munopositive astrocytes (Fig. 5C).

Acute T3 administration inducesthe expression of Shh mRNA inthe adult brain and corticalneurons in vitro

Next, we examined whether acute T3

treatment influences Shh expression invivo. Acute T3 administration resultedin a significant up-regulation of ShhmRNA in layer V of cortex, as well asthe lateral striatum of adult rats, sug-gesting a rapid transcriptional regula-tion of Shh mRNA by T3 (Fig. 6A). Toaddress direct effects of T3 on corticalneuronal Shh mRNA levels, we per-formed qPCR analysis on primary cor-tical neurons in vitro that were treatedwith 20 nM T3 for 3 h. Primary corticalcultures significantly up-regulatedShh mRNA in response to acute T3

treatment, supporting a direct effect ofT3 on Shh expression (Fig. 6C). Corticalneurons were found to express TRs invitro as confirmed by immunohisto-chemistry experiments (Fig. 6B).

Thyroid hormone treatment isassociated with enhanced histoneacetylation of the Shh promoter

To investigate whether the rapidtranscriptional up-regulation of Shh af-ter acute T3 treatment in vivo is associ-ated with enhanced histone acetylationat the Shh promoter, we carried outChIP assays for AcH3 and AcH4 within

upstream regions of the Shh gene in cortical tissue. AfterChIP, we performed qPCR analysis for upstream regionsof the Shh gene that contained putative thyroid hormoneresponse elements (TREs) (�6423 to �6413 bp and�6270 to �6261 bp) and also within the first 200 bp fromthe transcriptional start site. Our results revealed that neo-cortical tissue derived from T3-treated animals had signif-icantly enhanced acetylation of both histone H3 and H4 atputative TRE-containing regions upstream of the Shh geneand close to the transcriptional start site in the Shh pro-moter (Fig. 7, A and B).

We next examined whether acute T3 treatment influ-enced acetylation within the first 200 bp from the tran-

FIG. 4. Shh mRNA is significantly up-regulated in the adult rat brain in response to chronicT3 administration. Adult male rats received chronic T3 (Chr T3) administration for a period of10 d to render them hyperthyroid, and the levels of Shh, Ptc, and Smo mRNA weredetermined by in situ hybridization. Shown is a schematic representation of a coronal sectionof the adult rat brain with a color code depicting the regions quantitated (A). Also shown arerepresentative autoradiographs of Shh, Ptc, and Smo mRNA expression in coronal sections ofthe adult brains from vehicle and chronic T3-treated male rats (B). Adult-onsethyperthyroidism resulted in a robust and significant increase in Shh expression in the VDB,cortex, and Lat.St (C). Chronic T3 treatment significantly increased the expression of Ptc (D)and Smo (E) mRNA in the adult rat brain. Results are expressed as a percentage of controland are the mean � SEM (n � 5–6/group). *, P � 0.05 compared with control (Student’sunpaired t test). White bars, Vehicle-treated controls; hatched bars, chronic T3-treatedanimals.


scriptional start site of both the Ptc and Smo genes. Weobserved no change in the enrichment of AcH3 and AcH4within Ptc or Smo gene upstream regulatory regions.AcH3 at the Ptc and Smo promoter, fold change (Ptc):control, 1 � 0.39; T3 treated, 0.75 � 0.44; fold change(Smo): control, 1 � 0.24; T3 treated, 1.41 � 0.40. AcH4

at the Ptc and Smo promoter, fold change (Ptc): control,1 � 0.34; T3 treated, 1.31 � 0.52; fold change (Smo):control, 1 � 0.22; T3 treated, 0.97 � 0.35. Data are themean � SEM (P 0.05, Student’s t test). Taken together,these data indicate that the enhanced expression of ShhmRNA in the cortex is accompanied by significant in-creases in histone acetylation levels within Shh, but not Ptcor Smo, transcriptional regulatory sequences.

Discussion

Thyroid hormone is known to exert profound effects onneurodevelopment and retains a powerful influence on

FIG. 5. Subchronic T3 treatment enhances the number of �-galactosidase immunopositive cells within the neocortex of Shh�/LacZ

mice. Shown are representative images of �-galactosidase expressingcells in the cortex after short-duration T3 treatment over 2 d in Shh�/LacZ

mice. T3 treatment significantly increased the number of cells thatwere strongly immunopositive for �-galactosidase in layer V of cortex(A). Results are expressed as a percentage of vehicle-treated controland are the mean � SEM (n � 5/group). *, P � 0.05 compared withcontrol (Student’s unpaired t test). Shown is a schematicrepresentation (B) of a coronal section illustrating �-galactosidaseimmunopositive cells in Shh�/LacZ mice. The boxed area is enlarged toshow a representative area indicating the presence of several stronglyimmunopositive �-galactosidase immunopositive cells in the neocortexof T3-treated Shh�/LacZ mice. Double immunofluorescence experimentsrevealed the colocalization of �-galactosidase with the matureneuronal marker NeuN but not with the astroglial (GFAP) oroligodendrocytic (RIP or NG2) markers examined (C).

FIG. 6. Acute T3 treatment increases Shh mRNA expression in vivo andin vitro. Adult male rats received a single injection of T3, and the levelsof Shh mRNA were determined 3 h later by in situ hybridization. AcuteT3 treatment resulted in a significant increase in Shh mRNA expressionin the cortex and Lat.St (A). Results are expressed as a percentage ofcontrol and are the mean � SEM (n � 3–5/group). *, P � 0.05compared with vehicle-treated controls (Student’s unpaired t test).Ctx(V), Cortical layer V. Shh, Ptc, and Smo mRNA was determined byqPCR from T3-treated primary cortical neuron cultures, isolated frome17.5 pups that were grown in vitro for 9 d before acute treatmentwith 20 nM T3 for 3 h. Shown are representative images of MAP-2immunopositive primary cortical neurons expressing all TR isoforms (B)with their nuclei counterstained with Hoechst 33342. Acute T3

treatment of cortical cultures resulted in a significant increase in ShhmRNA expression, whereas Ptc and Smo mRNA remained unchangedas revealed by qPCR analysis (C). All genes were normalized to thehousekeeping gene Hprt. Results are expressed as fold change and arethe mean � SEM (n � 3–5/group). *, P � 0.05 compared with vehicle-treated controls (Student’s unpaired t test).


plasticity within the mature brain, including key effects onadult hippocampal neurogenesis (13, 15, 50, 51). Al-though several thyroid hormone-responsive genes, such asreelin, Dab-1, and Neurogenic differentiation, have beenhypothesized to mediate the neurodevelopmental influ-ences of thyroid hormone, most of these genes are tran-scriptionally responsive to thyroid hormone only duringselect developmental time windows (22, 52). In contrast,there are relatively few genes that retain thyroid hormoneresponsivity across the life span, for example neuroendo-crine specific protein and octamer transcription factor-1,which are reported to be regulated in both the embryonicand adult brain (24). Such thyroid hormone-responsivegenes are particularly interesting, because they are candi-dates to mediate the effects of thyroid hormone in both thedeveloping and mature brain. Although the effects of thy-roid hormone are spatiotemporally specific, it is quite pos-sible that the same sets of target genes may be redeployed

across the lifespan to mediate diverse developmental andneuroplastic consequences. We provide novel evidencethat the major developmental morphogen Shh is a thyroidhormone-responsive gene from the very earliest stages ofembryonic forebrain development into adulthood.

Perhaps the most dramatic example of the tissue mor-phoregulatory effects of thyroid hormone are those ob-served during amphibian metamorphosis, a process en-tirely dependent on the induction of a genetic program bythyroid hormone (53). Previous evidence indicates thatXhh is an early, direct target gene of thyroid hormone andis required to mediate the metamorphic effects of thyroidhormone in the gastrointestinal system (33). It is interest-ing to note that thyroid hormone-mediated induction ofXhh in tadpoles is tissue-specific with no effects observedin the brain (33). In contrast, we find that in the embryonicand adult mammalian brain, thyroid hormone perturba-tions induce a bidirectional regulation of the Shh signalingcascade in a region-specific manner. Although TRs areknown to be expressed in the fetal brain well before theinitiation of embryonic thyroid hormone synthesis (7, 9),and epidemiological evidence indicates that thyroid hor-mone perturbations in the first trimester influence fetalbrain development (1, 54), so far, only few thyroid hor-mone-responsive genes within the fetus have been iden-tified (55, 56). Our results indicate that maternal hy-perthyroidism significantly up-regulates embryonic Shhexpression in ventral forebrain signaling areas and thediencephalic signaling center of the Zli, and maternal hy-pothyroidism results in a region-specific decline of Shhwithin the embryonic Zli. This suggests that effective Shhsignaling from ventral forebrain signaling areas is likely tobe enhanced with maternal hyperthyroidism due to a ro-bust up-regulation of the ligand, and reduced with mater-nal hypothyroidism, due to down-regulation of the recep-tor complex as well as reduced ligand from the Zli.

Our results raise the possibility that specific neurode-velopmental actions of thyroid hormone may be mediatedby its control of the Shh pathway, providing a mechanisticlink between thyroid hormone perturbations and earlydevelopmental effects. For example, maternal thyroidhormone deficiency is associated with impaired corticalinterneuron development, in particular of parvalbuminand calretinin immunoreactive neocortical interneurons(10, 11, 57). It is particularly intriguing in this regard tonote that reduced Shh signaling in the embryonic ventralforebrain alters cortical interneuron composition, reduc-ing parvalbumin positive interneuron number (58).

Adult-onset perturbations in thyroid hormone statusalso significantly regulated the expression of Shh signalingcomponents in a bidirectional fashion, suggesting a con-tinued sensitivity of Shh expression to thyroid hormone

FIG. 7. Acute T3 treatment increases histone acetylation withinupstream regulatory regions of the Shh gene in the adult rat cortex.ChIP assays were performed for pan histone H3 and H4 acetylationchanges within gene regulatory sequences from �184 bp to thetranscriptional start site of the Shh gene. Cortical tissue derived fromT3-treated animals had significantly enhanced acetylation of histone H3and H4 (AcH3 and AcH4) close to the transcriptional start site withinthe Shh promoter after acute T3 treatment (A). ChIP analysis withinupstream regions of the Shh gene that contained putative TREs(�6554 to �6204 bp) revealed that cortical tissue derived from T3-treated animals had increased acetylation of both histone H3 and H4within these regulatory sequences as well (B). Results are expressed asfold change and are the mean � SEM (n � 7–10/group). *, P � 0.05compared with vehicle-treated controls (Student’s unpaired t test).


into adulthood. T3 administration resulted in enhancedShh mRNA levels within the adult neocortex, VDB, andlateral striatum, accompanied by significant increases inthe expression of both Ptc and Smo mRNA. Excess thyroidhormone levels also appear to unmask Smo expressionwithin the neocortex, a brain region that normally hasvery low levels of Smo mRNA in euthyroid controls, andthis could serve to potentiate the thyroid hormone-medi-ated effects on the Shh signaling pathway through in-creased expression of both the ligand as well as the sig-naling receptor. The enhanced Ptc expression observed inT3-treated adult animals could be interpreted as reflectiveof such increases in Shh signaling, because Ptc is a tran-scriptional target of Shh (59, 60). T3 administration alsosignificantly enhanced Smo mRNA within the hippocam-pal neurogenic niche. It is possible that enhanced Shh li-gand from the VDB may be transported via the septo-hippocampal pathway resulting in increased Shh signalingwithin the hippocampus (61), which is supported by theevidence of enhanced Ptc expression in the DG. In thiscontext, it is noteworthy that T3 administration is capableof increasing the postmitotic survival and neuronal dif-ferentiation of hippocampal progenitors (62), an effectthat overlaps with the enhanced survival observed aftertreatment with Smo agonists (63).

Although adult hyperthyroidism significantly en-hanced Shh ligand expression, we did not observe anychange in the ligand in adult-onset hypothyroid animalswithin the brain regions examined. It is possible to spec-ulate that despite reduced circulating thyroid hormonelevels, thyroid hormone availability in specific adult brainregions may not exhibit as steep a decline through themodulation of local deiodinase expression and activity. Itis possible that the relative extent of unliganded TR re-pressor effects on Shh mRNA expression may still be min-imal in the adult-onset hypothyroid brain. However, asingle previous study indicates that postnatal hypothy-roidism reduces Shh expression in the cerebellum, an effectsuggested to contribute to the cerebellar morphologicaldefects observed with postnatal hypothyroidism (64). Incontrast to the extensive effects of adult-onset hyperthy-roidism on expression of the Shh cascade, hypothyroidismin adulthood resulted in a spatially restricted and selectivedown-regulation of Smo expression within the DG. Adult-onset hypothyroidism is known to result in a neurogenicdecline, through a reduced proliferation, survival, andneuronal differentiation of hippocampal progenitors (12,13, 15). Shh is essential to the maintenance of the adulthippocampal stem cell niche and is known to modulate theproliferation, survival, and maintenance of adult progen-itors (29, 31). We have previously demonstrated an effectof adult-onset hypothyroidism on hippocampal progeni-

tors (13). Hypothyroidism resulted in a significant de-crease in Smo mRNA levels selectively in the DG, raisingthe intriguing possibility that the neurogenic decline ob-served in hypothyroid animals may be mediated througha reduced responsivity to Shh signaling.

Subchronic T3 treatment also enhanced the number of�-galactosidase immunopositive cells within the neocor-tex of Shh�/LacZ reporter mice, and colocalization exper-iments revealed that the expression of �-galactosidase wasneuronal. We did not observe �-galactosidase expressionin GFAP-positive astrocytes or NG2/RIP immunopositiveoligodendrocytes in the neocortex. Recent studies suggestthat thyroid hormone enhances oligodendrocytic progen-itor recruitment and maturation to promote recovery inanimal models of demyelination (65–67), and in somecases, it is accompanied by enhanced Shh expression (65).Further, under conditions of damage, reactive astrocyteswithin the cortex are reported to express Shh and induceoligodendrocytic progenitor proliferation (68). Shh ad-ministration into the neocortex of normal animals is alsocapable of recruiting premyelinating oligodendrocytic pro-genitors (30, 69). Our results indicate that T3 administrationin normal animals induces neuronal Shh expression, and thismay serve to recruit premyelinating oligodendrocytic pro-genitors even in the absence of overt damage.

The rapid effects of acute T3 administration on in-creased Shh expression in vivo and in cortical neurons invitro suggest that the regulation of Shh expression by thy-roid hormone in the brain may involve direct mechanisms.In contrast, the enhanced Smo and Ptc expression ob-served in adult-onset hyperthyroid animals is more likelyto be a secondary consequence of changes in expression ofthe ligand. In silico analysis indicated the presence of pu-tative TREs within upstream regulatory sequences of theShh gene. Thyroid hormone mediated transcriptional reg-ulation is associated with recruitment of coactivators andtargeted histone acetylation changes in upstream regionsof thyroid hormone-responsive genes (70). Previous stud-ies from Xenopus demonstrate that T3 bound to TRs canrecruit histone acetyl transferases to its target genes (71,72), and the expression of thyroid hormone-responsivegenes in Xenopus has also been shown to correlate withthe acetylation status of their promoters (73). Our resultsdemonstrate significant and rapid in vivo increases inglobal histone H3 and H4 acetylation both at the putativeTRE sites within upstream regions of the Shh gene, as wellas close to the transcriptional start site. Although evidenceof rapid increases in histone acetylation provides partialsupport to the notion that Shh is directly transcriptionallyregulated by thyroid hormone, future experiments are re-quired to identify promoter elements that mediate thyroidhormone responsivity of the Shh gene. Further experi-


ments to elucidate the role of select TR isoforms in thyroidhormone-mediated Shh transcriptional regulation, to ad-dress whether TRs exert their effects as homodimers orheterodimers with RXR, RAR, or TRAP, and to identifythe TREs or retinoic acid response elements that mediateTH responsivity are required to unequivocally demon-strate that Shh is a direct thyroid hormone target gene.

To the best of our knowledge, we provide the first ev-idence that Shh expression is robustly regulated by thyroidhormone in the brain. Recent elegant studies have dem-onstrated an opposite regulation, showing that Shh sig-naling can modulate thyroid hormone activity in neuronalcells through the regulation of deiodinases 2 and 3. Shh hasbeen reported to reduce thyroid hormone signalingthrough decreased deiodinase 2 and enhanced deiodinase3 activity in astrocytes and neurons, respectively (74). Thisthen evokes the tantalizing but speculative possibility of anegative feedback loop wherein thyroid hormone en-hances neuronal Shh signaling and Shh serves to restrictthyroid hormone-mediated effects via deiodinase activitymodulation. This raises the possibility of a powerful in-terplay between these two systems that may then play animportant role in neurodevelopment, adult plasticity, andpathophysiology.

In conclusion, we find that the Shh pathway displays astriking and selective regulation within the adult brain andin distinct signaling areas of the early embryonic forebrainunder conditions of adult-onset, and maternal, hyper- andhypothyroidism. We show that Shh expression is rapidlyregulated both in vivo and in vitro in response to thyroidhormone, accompanied by significant epigenetic histonemodifications within upstream regions of the Shh gene.Both thyroid hormone and Shh have been independentlydemonstrated to regulate diverse aspects of central ner-vous system development, including effects on prolifera-tion and maturation, and to retain a profound influence onthe adult brain, modulating recruitment, survival, and dif-ferentiation of neuronal and oligodendrocytic progenitors(13, 14, 29, 31). Our results suggest that specific devel-opmental and plasticity associated actions of thyroid hor-mone may be mediated by its control of the Shh pathwayand motivate future experiments to determine the mech-anistic contribution of Shh to the effects of thyroid hor-mone in both the developing and mature nervous system.

Acknowledgments

We thank S. Agashe and S. Banerjee for technical assistance.

Address all correspondence and requests for reprints to: Dr.Vidita A. Vaidya, Department of Biological Sciences, Tata In-stitute of Fundamental Research, Homi Bhabha Road, Mumbai400005, India. E-mail: [email protected].

This work was supported by intramural funds from Tata In-stitute of Fundamental Research and by Wellcome Trust SeniorOverseas Fellowships in Biomedical Sciences 04082003114133(to V.A.V.) and 056684/Z/99/Z (to S.T.).

Disclosure Summary: The authors have nothing to disclose.

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